Multi-resolution microscope reveals virus-cell processes in new detail
Top image: The method tracks the fast dynamics of a nanoparticle interacting with a cell, while observing the large-scale environment of the cell in high resolution. [Kevin Welsher, Princeton University]
Using a novel 3D multi-resolution microscope, researchers have imaged a virus-like particle attaching to, and penetrating a cell.
Based on two-photon laser-scanning microscopy (2P-LSM), the method can track the fast dynamics of a nanoparticle interacting with a cell, while observing the large-scale environment of the cell in high resolution.
According to Princeton University researchers, Kevin Welsher and Haw Yang, the method provides greater insight into particle-cell interactions than fluorescence and electron microscopy, single-particle tracking and super-resolution microscopy.
"The challenge in imaging these events is that viruses and nanoparticles are small and fast, while cells are relatively large and immobile,” says Welsher. “That has made it very hard to capture these interactions.”
To study cellular processes in detail, the researchers first developed a so-called target-locking implementation to track the fast dynamics of a nanoparticle interacting with a cell.
Combining a system of objective lenses, prism mirrors and avalanche photo detectors with a feedback mechanism, the researchers could lock a microscope objective focus onto a nanoparticle in real-time.
"Three dimensional target-locking is achieved using prism mirrors and an offset pinhole to detect [nanometre] deviations in a particle's X,Y and Z positions," explains Welsher. "These are used to move a 3D piezo-stage to counteract the particle's motion."
The set-up detects deviations in a nanoparticle's X,Y and Z positions. [Kevin Welsher, Princeton University]
The researchers then integrated this system with a modified two photon laser scanning microscope that would allow them to observe the lower resolution, larger-scale environment of the cell, at the same time.
To demonstrate their method, the researchers first created a virus-like particle by coating a polystyrene nanoparticle with fluorescent quantum dots, which was then studded with Tat peptide protein segments, derived from the HIV-1 virus.
This 3D movie shows footage of a virus-like particle (red dot) approaching a cell (green with reddish brown nucleus), as captured by Princeton University researchers Kevin Welsher and Haw Yang. The colour of the particle represents its speed, with red indicating rapid movement and blue indicating slower movement. Source: Nature Nanotechnology.
Particles were placed in a temperature controlled dish containing fibroblasts, ready for analysis.
As Welsher explains: "We observed a number of transient 'kiss and run' events where the [nanoparticle] probe transitioned from freely diffusing in the buffer to landing on the cell surface, traversing a plasma membrane before taking off and returning to free diffusion."
Statistical analyses of nanoparticle motion allowed diffusion paths to be quantified, and the researchers went onto colour code raw trajectories and generate dynamics heat maps of diffusion to characterise nanoparticle behaviour.
The researchers also used the 3D cellular contour data, provided by 2P-LSM, to further quantify nanoparticle transitions, correlating diffusivity with distance to a cell's surface.
"The possibilities enabled by this approach could pave the way to a better understanding of the longer-range interactions between ligands and receptors," says Welsher.
Thanks to their method's high time-definition and localisation, the researchers could also make use of a nanoparticle's motion across a cell surface to study 3D anatomical structures on the live cell membrane.
Combining this with dynamic heat mapping and the researchers were able to trace the complex nanoscale terrain features of a live cell, a feat simply not possible using conventional microscopy.
As Yang highlights, putting the two images together yielded a level of detail about the movement of nano-sized particles that had never before been achieved, Prior to this work, he said, the only way to see small objects at a similar resolution was to use electron microscopy, which requires killing the cell.
“What Welsher has done that is really different is that he can capture a three-dimensional view of a virus-sized particle attacking a living cell, whereas electron microscopy is in two-dimensions and on dead cells,” he adds. “This gives us a completely new level of understanding.”
The researchers believe the technology has potential benefits for both drug discovery and basic scientific discovery.
“We believe this will impact the study of how nanoparticles can deliver medicines to cells, potentially leading to some new lines of defence in antiviral therapies,” says Yang. “For basic research, there are a number of questions that can now be explored, such as how a cell surface receptor interacts with a viral particle or with a drug.”
Research is published in Nature Nanotechnology.
Read more at Princeton blogs.